Project Summary Aldosterone, a steroid hormone produced by the adrenal glomerulosa, plays a significant role in the pathophysiology of cardiovascular disease. Drugs that block aldosterone signaling are central to modern cardiovascular medicine; however, side effects restrict their use. Our understanding of aldosterone signaling at the molecular level is incomplete, limiting the development of new approaches to block aldosterone signaling. Aldosterone acts via the mineralocorticoid receptor (MR) to regulate transcription of genes encoding aldosterone- induced proteins. In the collecting ducts of the kidney, these proteins regulate activity of the epithelial sodium channel ENaC, resulting in increased channel activity and sodium retention. A number of aldosterone-induced proteins have been identified using cell culture models; however, deletion of these proteins in mice does not produce the expected phenotype of renal sodium wasting, elevated plasma potassium, and low blood pressure observed in mice with deletion of the MR specifically in the kidney epithelium (KS-MR-/- mice). Thus, aldosterone- induced proteins essential for activation of ENaC remain to be identified. This proposal seeks to identify these proteins using a novel approach that overcomes the limitations of previous screens. Specifically, this will be achieved by taking an in vivo approach that compares the transcriptional response to dietary sodium restriction, which stimulates endogenous aldosterone, in the collecting ducts of KS-MR-/- and control mice. Proteins encoded by transcripts that are induced in the control mice but not in the KS-MR-/- mice will be considered candidate proteins which will be further tested for aldosterone-dependence by Western blot and immunofluorescence. To determine whether candidate proteins modulate ENaC activity, amiloride-sensitive transepithelial current (a measurement for ENaC activity) will be determined in mouse kidney epithelial cells following siRNA knockdown of each candidate protein. The specific aims of this proposal are: (1) to identify novel aldosterone-induced proteins in the collecting duct and (2) to examine the role of aldosterone-induced proteins in activation of ENaC. This work will improve our understanding of aldosterone signaling and regulation of ENaC activity.